Table 5.

B-ALL Diagnostic Considerations and Ancillary Testing.

Clinical Information	Infant: B-ALL with KMT2A-r, NUTM1-r History of CML: absent in BCR::ABL1 + ALL-M Splenomegaly: favors chronic mveloid leukemia in ivmphoid blast crisis over BCR::ABL1+ ALL-M History of lymphoma: absent in B-ALL with MYC-r Eosinophilia: B-ALL with t(5:14): B-ALL, BCR::ABL1-like, ABL1 class-r (PDGFRB-r)
Immunopheno type	Flow cytometry With few exceptions generally not specific enough to be considered diagnostic, but may focus additional testing.	CD10: nonspecific, but characteristically dim/negative in B-ALL with KMT2A-r, ZNF384-r, CDX2/UBTF, MEF2D-r (MEF2D- r is also CD38++) Myeloid antigens (CD13/33): nonspecific, but typically positive in BCR::ABL1+ ALL-M and -L; B-ALL NUTM1-r, ZNF384-r, or ZNF384-like CD15:B-ALL with KMT2A-r or NUTM1-r (CD15 expression reported in a subset of cases [78]) CD2: B-ALL with DUX4-r (must be expressed with CD371, as CD2 expression by itself is non-specific) CD34: absent in B-ALL with t(1;19) and MYC-r Surface light chain (dim): non-specific: may be seen in B-ALL with MYC-r Markers that may not be available in all laboratories: Cytoplasmic μ chain: B-ALL with TCF3::PBX1, MEF2D-r, or CDX2/UBTF CRLF2: overexpressed in B-ALL, BCR::ABL1-like, IAK-STAT activated with CRLF-r CD24: absent in B-ALL with KMT2A-r CD371: B-ALL with DUX4-r (coexpressed with CD2) CD27+/CD44 dim or negative: B-ALL with t(12;21)(ETV6::RUNX1) and ETV6;;RUNX1-like CD9: strong expression in B-ALL with t(1;19)/TCF3::PBX1; absent in B-ALL with ETV6::RUNX1
	Immunohistochemistry (IHC)	TdT IHC: Differentiate between B-ALL (usually +) and aggressive mature B-cell lymphoma (rarely +) NUT IHC: Positive in B-ALL with NUTM1-r (consider NUT IHC in infant B-ALL if KMT2A-r negative) DUX4: Positive in B-ALL with DUX4-r
Cytogenetics	Karyotype	Modal chromosome number and/or DNA index: B-ALL, Hyperdiploid (>50 chromosomes) B-ALL, Hypodiploid: Near Haploid (24–31) and Low Hypodiploid (32–39 chromosomes; if low hypodiploid consider germline testing) Translocations (may require confirmation by FISH and/or molecular assays): t(9;22), KMT2A-r, t(5;14); t(1;19)
	Fluorescence in situ hybridization (FISH)	BCR::ABL1 fusion probe: BCR::ABL1+ALL-M will have BCR:ABL1+ granulocytes and may show a discrepancy between total % blasts and % bCr-ABL1+ cells ETV6::RUNX1 fusion probe: t(12;21)(ETV6::RUNX1); also identifies B-ALL with iAMP21. KMT2A breakapart: B-ALL with KMT2A-r TCF3: B-ALL with t(l;19) - confirm with karyotype/fusion studies If the above FISH studies are negative, then reflex to a BCR::ABL1-like panel to evaluate for rearrangements in: ABL1, ABL2, CSF1R, PDGFRB: B-ALL, BCR::ABL1-like, ABL1 class-r CRLF2, IAK2, EPOR: B-ALL, BCR::ABL1-like, IAK-STAT activated NTRK3: subset of B-ALL, BCR::ABL1-like, NOS (responds to TKIs) MYC breakapart: B-ALL with MYC-r (if +, consider adding BCL2 and BCL6 probes) Additional commercially available probes facilitate detection of B-ALL with: MEF2D::BCL9 fusion: MEF2D-r (subset) ZNF384 breakapart: ZNF384-r HLF breakapart: HLF-r NUTM1 breakapart: NUTM1-r
Molecular Diagnostics	Gene panel sequencing Often colloquially referred to as a “next generation sequencing (NGS) panel”	PAX5: B-ALL with PAX5 P80R IKZF1: B-ALL with IKZFl N159Y Other targetable kinases and important co-mutations (e.g. TP53, IKZF1)
	Whole Genome Sequencing: DNA based genomic analysis	Aneuploidy Rearrangements, including those that do not result in a chimeric transcript Mutations
	Targeted molecular assays: RT-PCR using sequence-specific primers for each partner in a known fusion protein	BCR::ABL1 (p190 versus p210; may be used for MRD): BCR::ABL+ ALL-M and -L UBTF::ATXN7L3: CDX2/UBTF-deregulated Identification of mutations that may be cryptic on cytogenetic studies: t(l2;2l)(ETV6::RUNX1) t(l;l9)TCF3::PBX1 Rapid detection of specific mutations in order to guide early therapy.
	Targeted transcriptome sequencing: RNA-Seq using specific primers. For fusion transcript analysis, primers are utilized for one member of the fusion, and anchored multiplex PCR captures the full array of in-frame fusion partners.	BCR and ABL1: BCR::ABL1+ ALL-M and - L; subset of B-ALL, BCR::ABL1-like KMT2A: B-ALL with KMT2A-r ETV6 and RUNX1: B-ALL with t(12;21)(ETV6::RUNX1) TCF3: B-ALL with TCF3::PBX1, TCF3::HLF, TCF3-ZNF384 MYC: B-ALL with MYC-r (if +, then evaluate for BCL2-r, BCL6-r) ABL1, ABL2, CSF1R, PDGFRB: B-ALL, BCR::ABL1-like, ABL1 class-r CRLF2, IAK2, EPOR: B-ALL, BCR::ABL1-like, IAK-STAT activated FLT3, FGFR1, NTRK3, PTK2B: B-ALL, BCR::ABL1-like. NOS The following fusions may not yet be available on standard gene fusion panels: ZNF384: ZNF384-r UBTF::ATXN7L3: CDX2/UBTF-deregulated Evaluation for selected single gene mutations and gene expression is also possible via targeted transcriptome sequencing, and may be incorporated into panels
	Whole Transcriptome Sequencing: RNA-Seq using random primers to evaluate the full transcriptome	Aneuploidy, partial gains/loss of chromosomes Identification of gene rearrangements that generate fusion proteins Mutations in transcribed sequences Gene expression (B-ALL, BCR::ABL1-like; PAX5alt; ETV6::RUNX1-like)
	Somatic Hypermutation	Negative: B-ALL with MYC-r Positive: MYC-r aggressive B-cell lymphomas
	Clonality studies	Recommended at diagnosis to facilitate molecular MRD monitoring *The presence or absence of IGH and/or TCR clones cannot utilized for lineage assignment (B-ALL versus T-ALL).

New Entities in the ICC subclassification are underlined.